HIV vaccination compositions comprising vaccinia VLPS and plant-produced VLPS presenting HIV antigens

Abstract

Disclosed are compositions for generating an immune response against human immunodeficiency virus (HIV) and their methods of uses.

We claim: 
     
       1. A method of generating an immune response in a mammalian subject against HIV comprising:
 administering a vaccinia virus-like particle (VLP) to the mammalian subject, wherein the vaccinia VLP presents HIV Gag, a fragment of gp41, or both; and 
 administering a plant-produced HIV VLP to the mammalian subject, wherein the plant-produced HIV VLP presents HIV Gag and a fragment of gp41 and is isolated from plant tissue transformed with a dual replicon geminivirus-based plant expression vector comprising a T-DNA region that comprises:
 a first nucleic acid sequence encoding Gag and a first promoter region upstream of the first nucleic acid sequence encoding Gag; and 
 a second nucleic acid sequence encoding a fragment of gp41 and a second promoter upstream of the second nucleic acid sequence encoding a fragment of gp41, 
 
 wherein the mammalian subject is administered the vaccinia VLP and the plant-produced HIV VLP in an amount sufficient to generate an HIV immune response in the mammalian subject. 
 
     
     
       2. The method of  claim 1 , wherein the molar ratio of HIV Gag and the fragment of gp41 expressed by the plant produced HIV VLP is 1.7 to 11.8. 
     
     
       3. The method of  claim 1 , wherein the fragment of gp41 is dgp41. 
     
     
       4. The method of  claim 1 , wherein the vaccinia VLP administered presents both Gag and dgp41. 
     
     
       5. The method of  claim 1 , wherein the mammalian subject is administered the vaccinia VLP at least 30 days prior to the administration of the plant-produced HIV VLP. 
     
     
       6. The method of  claim 1  further comprising administering to the mammalian subject a second dose of the plant-produced HIV VLP. 
     
     
       7. The method of  claim 1 , wherein the vaccinia VLP is isolated from a mammalian cell transfected with at least one replicating but highly attenuated vaccinia virus vector selected from the group consisting of:
 a first replicating but highly attenuated vaccinia virus vector comprising a third nucleic acid sequence encoding Gag, 
 a second replicating but highly attenuated vaccinia virus vector comprising a fourth nucleic acid sequence encoding a fragment of gp41, and 
 a third replicating but highly attenuated vaccinia virus vector comprising the third nucleic acid sequence encoding Gag and the fourth nucleic acid sequence encoding fragment of gp41. 
 
     
     
       8. The method of  claim 7 , wherein the replicating but highly attenuated vaccinia virus vector is NYVAC. 
     
     
       9. The method of  claim 1 , wherein the T-DNA region of the geminivirus-based vector plant expression vector further comprises a nucleic acid sequence encoding at least one replication gene. 
     
     
       10. The composition of  claim 9 , wherein the geminivirus-based vector is a bean yellow mosaic virus-based vector, the nucleic acid sequence encoding at least one replication gene that encodes Rep/RepA. 
     
     
       11. The method of  claim 10 , wherein the first promoter region and the second promoter region comprise a nucleic acid sequence encoding the cauliflower mosaic virus 35S promoter (P35). 
     
     
       12. The method of  claim 11 , wherein the first promoter region and the second promoter region further comprise two translation enhancer binding sites downstream of nucleic acid sequence encoding P35. 
     
     
       13. The method of  claim 12 , wherein the T-DNA region of the plant expression vector further comprises a pair of long intergenic regions, wherein the pair of long intergenic regions flank a portion of the T-DNA region that does not comprise the nucleic acid sequence encoding the silencing suppressor protein and does comprise the at least one replication gene, the first nucleic acid sequence encoding Gag and the first promoter region upstream of the first nucleic acid sequence encoding Gag; and/or the second nucleic acid sequence encoding a fragment of gp41 and a second promoter upstream of the second nucleic acid sequence encoding a fragment of gp41. 
     
     
       14. The method of  claim 13 , wherein the second nucleic acid sequence encoding the fragment of gp41 comprises the nucleic acid sequence of dgp41. 
     
     
       15. The method of  claim 9 , wherein the T-DNA region of the plant expression vector further comprises a nucleic acid sequence encoding a silencing suppressor protein, wherein the nucleic acid sequence encoding the silencing suppressor protein is upstream of the first nucleic acid sequence encoding Gag and the first promoter region upstream of the first nucleic acid sequence encoding Gag and upstream of the second nucleic acid sequence encoding a fragment of gp41 and a second promoter upstream of the second nucleic acid sequence encoding a fragment of gp41. 
     
     
       16. The method of  claim 9 , wherein the T-DNA region of the plant expression vector further comprises a nucleic acid sequence encoding barley α-amylase signal peptide, wherein the nucleic acid sequence encoding barley α-amylase signal peptide is upstream of the second nucleic acid sequence encoding a fragment of gp41 and downstream of the second promoter region. 
     
     
       17. A method of generating an HIV immune response in a mammalian subject, the method comprising:
 administering a vaccinia virus-like particle (VLP) to the mammalian subject, wherein the vaccinia VLP presents HIV Gag and a fragment of gp41; and 
 administering a plant-produced HIV VLP to the mammalian subject, wherein the plant-produced HIV VLP presents HIV Gag and a fragment of gp41 and is isolated from plant tissue transformed with a geminivirus-based plant expression vector, 
 wherein the vaccinia VLP is isolated from a mammalian cell transfected with at least one replicating but highly attenuated vaccinia virus vector and the mammalian subject is administered the vaccinia VLP and the plant-produced HIV VLP in an amount sufficient to generate an HIV immune response in the mammalian subject. 
 
     
     
       18. The method of  claim 17 , wherein
 the geminivirus-based plant expression vector comprises a T-DNA region comprising:
 a first nucleic acid sequence encoding Gag and a first promoter region upstream of the first nucleic acid sequence encoding Gag; and 
 a second nucleic acid sequence encoding a fragment of gp41 and a second promoter upstream of the second nucleic acid sequence encoding a fragment of gp41; and 
 
 the at least one replicating but highly attenuated vaccinia virus vector is selected from the group consisting of:
 a first replicating but highly attenuated vaccinia virus vector comprising a third nucleic acid sequence encoding Gag, 
 a second replicating but highly attenuated vaccinia virus vector comprising a fourth nucleic acid sequence encoding a fragment of gp41, and 
 a third replicating but highly attenuated vaccinia virus vector comprising the third nucleic acid sequence encoding Gag and the fourth nucleic acid sequence encoding fragment of gp41. 
 
 
     
     
       19. The method of  claim 1 , wherein the vaccinia VLP is administered to the mammalian subject first and the plant-produced HIV VLP is administered to the mammalian subject second.